Recombinant proteins of parapoxvirus ovis and pharmaceutical compositions therefrom

ABSTRACT

The invention relates to polynucleotides coding for the PPVO viral genome, to fragments of the polynucleotides coding for the PPVO genome and to polynucleotides coding for individual open reading frames (ORFs) of the PPVO viral genome. The invention also relates to recombinant proteins expressed from the above mentioned polynucleotides and to fragments of said recombinant proteins, and to the use of said recombinant proteins or fragments for the preparation of pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/717,640, filed 17 Dec. 2012, now allowed, which is a continuation ofU.S. patent application Ser. No. 10/481,112, filed 11 Jun. 2004, nowpatented as U.S. Pat. No. 8,357,363, which is a U.S. National PhaseApplication of International Patent Application No. PCT/EP2002/006440,having an international filing date of 12 Jun. 2002, which claimspriority to New Zealand Patent Application No. 512341 filed 13 Jun.2001. The contents of each of these applications are incorporated hereinby reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 595282000303SeqList.txt,date recorded: 5 Sep. 2014, size: 234,776 bytes).

FIELD OF THE INVENTION

The present invention relates to polynucleotides and recombinantproteins of Parapoxvirus ovis (PPVO) and their use, alone or incombination with other substances, for the manufacture of pharmaceuticalcompositions.

BACKGROUND OF THE INVENTION

It is known that latent and chronically persistent viral infections canbe activated or reactivated by immunosuppression, or conversely that theimmune system suppresses acute diseases which may be caused by a latentvirus (for example a latent herpes virus infection recurs as a result ofimmunosuppression in the form of lip vesicles in cases of stress or theadministration of cortisone). It is also known that chronicallypersistent latent viral infections can only be treated with difficultyor not at all using conventional low-molecular-weight antiviralsubstances.

It was demonstrated that class I restricted cytotoxic T cells werecapable of inhibiting hepatocellular HBV gene expression inHBV-transgenic mice, and that this process was caused by TNF-α andIFN-γ.

It is also known that in the case of chronically persistent viralinfections a superinfection with another virus can produce antiviraleffects against the chronically persistent virus. The dependence of thiseffect on interferons such as IFN-γ, as well as other cytokines andchemokines, such as TNF-α, which are secreted by T cells, natural killercells and macrophages, has been demonstrated.

BAYPAMUN®, a pharmaceutical product for inducing “paraspecificimmunity”, i.e., a pharmaceutical product for inducing the unspecificimmune system, is used therapeutically, metaphylactically andprophylactically for the treatment of animals in need. BAYPAMUN® ismanufactured from chemically inactivated PPVO strain D1701 (see GermanPatent DE3504940). The inactivated PPVO induces in animals non-specificprotection against infections with the most diverse types of pathogens.It is assumed that this protection is mediated via various mechanisms inthe body's own defense system. These mechanisms include the induction ofinterferons, the activation of natural killer cells, the induction of“colony-stimulating activity” (CSA) and the stimulation of lymphocyteproliferation. Earlier investigations of the mechanism of actiondemonstrated the stimulation of interleukin-2 and interferon-α.

The processes for the production of the above-mentioned pharmaceuticalcompositions are based on the replication of the virus in cultures ofsuitable host cells.

One aspect of the invention relates to the use of particle-likestructures comprising recombinant proteins of the invention. Theseparticle-like structures can be, e.g., fusion proteins, protein-coatedparticles or virus-like particles.

Methods to produce fusion proteins, protein-coated particles orvirus-like particles comprising recombinant proteins of the inventionare well known to persons skilled in the art: Casal (Biotechnol. Genet.Eng. Rev. (2001) 18:73-87) describes the use of baculovirus expressionsystems for the generation of virus-like particles. Ellis (Curr. Opin.Biotechnol. (1996) 7(6):646-652) presents methods to produce virus-likeparticles and the application of suitable adjuvants. Roy (Intervirology(1996) 39(1-2):62-71) presents genetically engineered particulatevirus-like structures and their use as vaccine delivery systems. Methodsto produce fusion proteins are also well known to the person skilled inthe art (Gaudin, et al., Gen. Virol. (1995) 76:1541-1556; Hughson, Curr.Biol. (1995) 5(3):365-374; Uhlen, et al., Curr. Opin. Biotechnol. (1992)3(4):363-369). Known to the person skilled in the art is also thepreparation of protein-coated micro- and nanospheres (Arshady,Biomaterials (1993) 14(1):5-15). Proteins can be attached tobiodegradable microspheres (Cleland, Pharm Biotechnol. (1997) 10:1-43)or attached to other polymer microspheres (Hanes, et al., Pharm.Biotechnol. (1995) 6:389-412) such as, e.g., polysaccharides (Janes, etal., Adv. Drug Deliv. Rev. (2001) 47(1):83-97).

PPVO NZ2 is another Parapoxvirus strain that exhibits immunostimulatoryeffects when administered in inactivated form to mammals.

The closest prior art describes the construction of an expressionlibrary representing about 95% of the PPVO NZ2 genome using the Vaccinalister virus to create recombinant viruses comprising the completeVaccina lister genome and various fragments of the PPVO genome (Mercer,et al., Virology, (1997) 229:193-200). For the construction of thelibrary, 16 PPVO DNA fragments with an average size of 11.4 kb wereinserted into the Vaccinia lister genome. Each fragment was mappedrelative to the PPVO restriction endonuclease maps but was otherwiseuncharacterized (FIG. 1). It was found that a major portion of the PPVOgenes were expressed in cells infected by the recombinant virus. Theauthors also showed that the entirety of all PPVO proteins expressed bysome of the recombinant viruses of the expression library was able toprovide protection against challenge with virulent PPVO. Expression ofPPVO genes of the individual recombinant viruses has been demonstratedby immunofluorescence and immune precipitation (Mercer, et al., Virology(1997) 229:193-200).

To identify components of PPVO responsible for the vaccinating activityof PPVO, the Vaccinia lister/PPVO NZ2 expression library was applied.

Based on the above background it was desirable to develop PPVO basedpharmaceutical compositions with antiviral and anti-tumor efficacy aswell as with efficacy in paraimmunization and other desirabletherapeutic effects. It was also desirable to obtain a pharmaceuticalcomposition that exerts its full therapeutic effect while showing fewerside effects. It was furthermore desirable to find methods to producePPVO based pharmaceutical compositions in large quantities and ineconomically advantageous manners.

These desirable effects have been achieved by the systematic use ofselected recombinant proteins of PPVO alone or in combination with otherrecombinant proteins from PPVO for the preparation of pharmaceuticalcompositions for the treatment of objects in need.

SUMMARY OF THE INVENTION

The invention relates to polynucleotides coding for the PPVO viralgenome, to fragments of the polynucleotides coding for the PPVO genomeand to polynucleotides coding for individual open reading frames (ORFs)of the PPVO viral genome. The invention also relates to fragments ofsaid polynucleotides of at least 15 or 30 or 100 base pairs in length.The invention also relates to recombinant proteins expressed from theabove mentioned polynucleotides and to fragments of said recombinantproteins of at least 5 or 10 or 30 amino acids, and to the use ofrecombinant proteins or fragments for the preparation of pharmaceuticalcompositions.

“Fragments” of a polynucleotide, within the meaning of the invention,shall be understood as polynucleotides that have the same nucleotidesequence as contiguous parts of the full length (the original)polynucleotide.

“Active fragments”, within the meaning of the invention, shall be thosefragments of the PPVO genome the expression products of which havedemonstrated to be pharmacologically active according to the invention,when inserted into the Vaccina lister genome and expressed in a suitablehost.

Whereas the use of the complete PPVO virus for the manufacture ofvaccines against PPVO challenge has been described, the presentinvention relates to the use of polynucleotides coding for the PPVOviral genome and selected fragments of the PPVO viral genome and ofselected PPVO expression products, alone or in combination with others,for the preparation of improved pharmaceutical compositions for thetreatment of various diseases.

The systematic use of selected genomic fragments of PPVO and theirrecombinant expression products makes it possible to producepharmaceutical compositions which contain fewer (and may not containany) inactive components (i.e., polynucleotides and proteins of PPVO) inaddition to the active components.

These pharmaceutical compositions which contain less, or do not containany additional inactive components are generally preferred by doctorsand patients compared to the less well defined biological preparationsof inactivated virus material. Furthermore, the possibility of producingthe recombinant product in fermentation processes allows an economicallyadvantageous mode of production. It is well known to persons skilled inthe art that an economically advantageous mode of production can beachieved, e.g., by using rapidly growing production organisms (hostorganisms) which might also place low demands on the culture mediumemployed. Microorganisms which can advantageously be used as hosts forthe production of recombinant proteins include, e.g., but are notlimited to, Escherichia coli, Bacillus spec., Corynebacterium spec.,Streptomyces spec., as well as yeasts, e.g., Saccharomyces cerevisiae,Candida spec., Pichia spec., Hansenula spec., and filamentous fungi,e.g., Aspergillus spec., Penicillium spec. and other suitablemicroorganisms.

Recombinant proteins of the invention can also be produced from celllines expressing the proteins of interest. These cell lines can berecombinant mammalian cell lines, recombinant insect cell lines (e.g.,using the baculovirus transfection system) or other suitable expressionsystems. Transfection can be achieved by various techniques known to theskilled person, one of which is the use or recombinant viruses such asthe Vaccinia virus/PPVO recombinants (VVOVs) described in the examples.

DESCRIPTION OF THE INVENTION

The invention relates to fragments of the PPVO genome of at least 15 or30 or 100 base pairs in length, and recombinant proteins expressedtherefrom and to the use of said fragments and recombinant proteins forthe preparation of pharmaceutical compositions. The invention alsorelates to individual genes (ORFs) of PPVO and their expressionproducts, and their use, alone or in combination with others, for thepreparation of pharmaceutical compositions.

A protein, within the meaning of the invention, is any polypeptide of atleast five amino acids. A recombinant protein, within the meaning of theinvention, is any protein that is expressed in a cell, to which thecoding polynucleotide was introduced using recombinant DNA technology.

A polynucleotide, within the meaning of the invention, is meant tocomprise, polyribonucleotides and/or polydesoxyribonucleotides.

Pharmaceutical compositions of the invention can be used asimmunotherapeutic or immunoprophylactic agents for the treatment ofinfectious and non-infectious immunodeficiencies. They can also be usedfor the treatment of tumor diseases, cancer, viral infections anddiseases associated therewith, such as, e.g., hepatitis, papillomatosis,herpes virus infections, liver fibrosis, for the prevention orprophylaxis of infectious diseases after stress (e.g., operations), forthe prevention and prophylaxis of infectious diseases by administrationprior to operations or procedures (e.g., preceding implantations ofartificial limbs or dental procedures), for the prophylactic andmetaphylactic treatment of non-viral infections, for the healing ofwounds, and in particular for accelerating wound-healing processes andfor promoting the healing of poorly healing or non-healing wounds (e.g.,Ulcus cruris), for diseases such as multiple sclerosis, warts and otherskin neoplasms, for allergic diseases, for preventing the onset ofsystemic allergies and for topical allergies and for improvingwell-being, e.g., in old age, for autoimmune diseases, chronicinflammatory diseases, such as, e.g., Crohn's disease, COPD and asthma.It is an object of the invention to use of polynucleotides andrecombinant proteins of PPVO for the production of pharmaceuticalcompositions for the treatment of the above mentioned conditions anddiseases in humans and animals.

The viral strains of the invention are PPVO NZ2 and homologues, such asD1701, NZ7, NZ10 and orf-11 strains. It is also possible to usepolynucleotides and recombinant proteins of the progeny of these strainsobtained by passaging and/or adaptation using specific cells, such as,e.g., WI-38, MRC-5 or Vero cells.

We have found that the identified recombinant proteins are effective forthe treatment of viral diseases, cancer and other diseases or conditionsin which a Th1 type immune response is of benefit. The results obtainedalso imply that PPVO gene products or parts thereof protect hepatitisvirus-expressing hepatocytes (e.g., hepatitis B virus, HBV, or hepatitisC virus, HCV) from immune attack through HBV or HCV specific cytotoxicCD8+ T cells circulating in the blood because T cells will not leave theblood stream if their specific antigen is not presented by liver sinusendothelial cells (LSEC, that anatomically separate hepatocytes from Tcells passing the liver with the blood). Therefore, we expect to have arecombinant protein that is derived from the ORFs 120-R3 (base pairs122616-136025 Bp, recombinant virus VVOV82) that is able todown-modulate or prevent side effects such as necroinflammatory liverdisease when immunostimulants, e.g., cytokines or any others includingthe proteins described above administered to, e.g., hepatitis patients.

Considering the knowledge about the influence of a Th1 type immuneinduction in conditions and diseases such as latent and or chronic viralinfections, proliferative diseases such as cancer and the capability ofrecombinant proteins that contain gene products of PPVO or parts thereofto induce a Th1 immune response or a local immune response selectively,we claim the use of polynucleotides and recombinant polypeptides of PPVOand recombinant proteins that contain gene products of PPVO or partsthereof for the manufacture of pharmaceutical compositions for use inhumans and animals. The recombinant proteins are made from products orparts thereof of the following open reading frames (ORFs) of PPVO NZ2:64r-96r (recombinants VVOV 285 and VVOV 330 as well as VVOV 243 and VVOV283), 18r-57 (recombinants VVOV 97, VVOV 96 and VVOV 245), 4r-14r(recombinant VVOV 215). The recombinant protein may also be made fromgene products or parts thereof of ORFs 120-R3 (recombinant VVOV 82). Theproteins may be prepared and used in any combination.

Recombinant proteins of PPVO within the meaning of the invention shallbe understood as proteins that derive from PPVO and are expressed inhomologous or heterologous systems other than the systems in which PPVOis naturally produced. Examples for recombinant proteins of PPVO areproteins of PPVO which are expressed using Vaccinia virus vectors andfibroblasts as host cells or baculovirus vectors and insect cells ashost cells. Recombinant proteins, within the meaning of the invention,could also be produced in bacterial cells (e.g., Escherichia coli,Bacillus spec., Streptomyces spec.) or in yeast (e.g., Saccharomycescerevisiae, Candida spec., Pichia pastoris, Hansenula spec.) systems. Inthese cases, polynucleotides of the PPVO genome would typically bebrought into the respective host genome so that PPVO genes are expressedby the host. Recombinant proteins of PPVO could also be expressed by theobject in need in the sense of a gene therapy.

Recombinant proteins, within the meaning of the invention, could also berecombinant virus particles that contain PPVO derived proteins.Recombinant proteins, within the meaning of the invention, could also bein form of viral-like particles that are formed or assembled from PPVOderived proteins. Recombinant proteins, within the meaning of theinvention, could also be chimeric proteins that contain PPVO geneproducts.

In a preferred embodiment of the invention the recombinant proteins areattached to particle-like structures or be part of particle-likestructures.

In another preferred embodiment of the invention the recombinantproteins are attached to, or part of, fusion proteins.

In another preferred embodiment of the invention the recombinantproteins are attached to, or part of, protein-coated particles.

In another preferred embodiment of the invention the recombinantproteins are attached to, or part of, virus-like particles.

Particle-like structures, such as particle-like fusion proteins,protein-coated particles or virus-like particles can be phagocytosed andprocessed by monocytes or macrophages. The process of phagocytosisenhances the efficacy of recombinant proteins of the invention in useswithin the meaning of the invention.

A particle-like structure, within the meaning of the invention, isparticulate matter in particle-like form of which the average particlesize and other characteristics are suitable for medical application.Preferred particle-like structures are, e.g., fusion proteins,protein-coated particles, or virus-like particles.

Immunomodulating activity is defined as local or systemic suppressionand/or stimulation and/or induction of any Th-1 or Th-2 type cytokineresponse or of any effector function of these cytokines, (e.g.,cytolytic or antiviral activity or humoral response) or the modulationof MHC cross-presentation. Immunomodulating activity could also be theinduction of apoptosis in antigen presenting cells or recruiting ofantigen presenting cells.

Nucleotides and recombinant proteins of the invention can beadministered at the same time or sequentially, administered with otheragents and drugs, e.g., with drugs that treat the disease or aresupportive, e.g., in the case of cancer therapy with antineoplastic orother anti-cancer agents or/and anti-coagulants or vitamins, pain reliefand others.

The nucleotides and recombinant proteins can be administeredsystemically (e.g., intravenously, subcutaneously, intramuscularly,intracutaneously, intraperitoneally), locally (e.g., into a tumor) ororally (per os). The recombinant proteins or products thereof should beformulated appropriately, e.g., in a non-pyrogenic solution orsuspension for i.v. use or in capsules for implantation or in capsulesfor per os use. Pharmaceutical compositions of the invention can beadministered, e.g., oral, nasal, anal, vaginal etc., as well asparenteral administration. Pharmaceutical compositions of the inventioncan be in the form of suspensions, solutions, syrups, elixirs orappropriate formulations in polymers as well as liposomes.

Recombinant proteins of the invention can also be prepared with suitablerecombinant cell lines and other cell lines. Alternatively,non-recombinant cell lines, such as WI-38, MRC-5, Vero cells could beinfected with recombinant viruses that carry the recombinant genes usingviral vectors such as, but not limited to, the Vaccina virus (e.g.,Vaccina lister). In addition, other suitable viruses can be used incombination with other suitable cells (e.g., using Vaccinia virusvectors and fibroblasts as host cells or baculovirus vectors and insectcells as host cells). It is advantageous to cultivate the recombinantcell cultures in high-cell-density fermentations to achieve favorableproductivity and a good overall process performance.

The invention relates to purified and isolated polynucleotides with thesequence of SEQ ID NO:1. The invention also relates to purified andisolated polynucleotides of at least 15 or 30 or 100 nucleotides whichbind under stringent conditions to the polynucleotide of SEQ ID NO:1 orits complementary sequences.

Stringent conditions, within the meaning of the invention are 65° C. ina buffer containing 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% (w/v) SDS.

The invention also relates to purified and isolated polynucleotideswhich comprise the polynucleotide sequence of SEQ ID NO:1 orpolynucleotide sequences encoding the same amino acid sequence andfragments of at least 15 or 30 or 100 nucleotides thereof. The inventionalso relates to recombinant proteins of five and more amino acidsencoded by these polynucleotides.

The invention also relates to purified and isolated polynucleotideswhich show at least 99%, 95% or 90% or 80% sequence homology to thepolynucleotides of the previous paragraph.

Homology of biological sequences, within the meaning of the invention,shall be understood as the homology between two biological sequences ascalculated by the algorithm of Needleman and Wunsch. (J. Mol. Biol.(1970) 48:443-453) using the BLOSUM62 substitution matrix (Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA (1992) 89:10915-10919) for proteinsand penalties of +4 and −3 for identical and non-identical bases,respectively, when comparing polynucleotide sequences. For comparison ofprotein sequences the gap creation penalty and the gap extension penaltyare 8 and 2, respectively. For comparison of polynucleotide sequencesthe gap creation penalty and the gap extension penalty are 20 and 3,respectively.

The invention also relates to purified and isolated polynucleotideswhich are active fragments of the PPVO genome, with a sequence selectedfrom a group of sequences consisting of nucleotides 122616-136025 of SEQID NO:1 (PPVO insert of VVOV 82), 31003-46845 of SEQ ID NO:1 (PPVOinsert of VVOV 96), 24056-33789 of SEQ ID NO:1 (PPVO insert of VVOV 97),10264-20003 of SEQ ID NO:1 (PPVO insert of VVOV 215), 82324-92502 of SEQID NO:1 (PPVO insert of VVOV 243), 47952-66263 of SEQ ID NO:1 (PPVOinsert of VVOV 245), 89400-103483 of SEQ ID NO:1 (PPVO insert of VVOV283), 74804-88576 of SEQ ID NO:1 (PPVO insert of VVOV 285), and102490-108393 of SEQ ID NO:11 (PPVO insert of VVOV 330).

The invention also relates to purified and isolated polynucleotide whichencode for the same amino acid sequence as the active fragments of thePPVO genome of the previous paragraph and to polynucleotides of at least15 or 30 or 100 nucleotides binding under stringent conditions to theabove mentioned active fragments of the PPVO genome or its complementarysequence.

The invention also relates to polynucleotides with 99%, 95%, or 90%, or80% sequence homology to sequences consisting of nucleotides122616-136025 of SEQ ID NO:1 (PPVO insert of VVOV 82), 31003-46845 ofSEQ ID NO:1 (PPVO insert of VVOV 96), 24056-33789 of SEQ ID NO:1 (PPVOinsert of VVOV 97), 10264-20003 of SEQ ID NO:1 (PPVO insert of VVOV215), 82324-92502 of SEQ ID NO:1 (PPVO insert of VVOV 243), 47952-66263of SEQ ID NO:11 (PPVO insert of VVOV 245), 89400-103483 of SEQ ID NO:1(PPVO insert of VVOV 283), 74804-88576 of SEQ ID NO:11 (PPVO insert ofVVOV 285), and 102490-108393 of SEQ ID NO:11 (PPVO insert of VVOV 330)or the respective complementary sequences.

The invention also relates to purified and isolated polynucleotide, witha sequence of nucleotides 3 to 539 (ORF L1), 781 to 449 (ORF L2r), 1933to 1664 (ORF L3r), 3269 to 2790 (ORF L4r), 2799 to 3851 (ORF L5), 2962to 3753 (ORF L6), 3784 to 3122 (ORF L7r), 4341 to 4129 (ORF L8r), 4904to 4428 (ORF 1ar), 6517 to 4970 (ORF 1r), 8042 to 6684 (ORF 2r), 9989 to8070 (ORF 3r), 11195 to 10062 ORF 4r), 11493 to 11227 (ORF 5r), 11802 to12038 (ORF 6), 12358 to 12080 (ORF 7r), 13980 to 12364 (ORF 8r), 14826to 14053 (ORF 9ar), 15080 to 15394 (ORF 10), 16838 to 15423 (ORF 11r),19021 to 16847 (ORF 12r), 19704 to 19156 (ORF 13r), 20314 to 19736 (ORF14r), 20401 to 22101 (ORF 15), 22125 to 22940 (ORF 6), 23003 to 23866(ORF 17), 26908 to 23873 (ORF 18r), 26926 to 27213 (ORF 19), 27626 to27216 (ORF 20r), 29754 to 27616 (ORF 21r), 32217 to 29800 (ORF 22r),33380 to 32418 (ORF 23r), 33602 to 33393 (ORF 24r), 34466 to 33612 (ORF25r), 34735 to 34502 (ORF 26r), 35905 to 34739 (ORF 27r), 37194 to 35905(ORF 28r), 37200 to 39248 (ORF 29), 41037 to 39229 (ORF 30r), 41374 to42066 (ORF 31), 42336 to 41731 (ORF 32r), 42407 to 41997 (ORF 33r),42410 to 43765 (ORF 34), 43770 to 43958 (ORF 35), 43980 to 44534 (ORF36), 45727 to 44537 (ORF 37r), 45760 to 46557 (ORF 38), 46567 to 47568(ORF 39), 47572 to 48303 (ORF 40), 48352 to 48621 (ORF 41), 49887 to48634 (ORF 42r), 49917 to 50693 (ORF 43), 50719 to 51102 (ORF 44), 51059to 51511 (ORF 44a), 51584 to 52591 (ORF 45), 52509 to 53066 (ORF 46),53523 to 53023 (ORF 47r), 53607 to 57473 (ORF 48), 58070 to 57528 (ORF49r), 57700 to 58662 (ORF 50), 59674 to 58673 (ORF 51r), 62089 to 59678(ORF 52r), 62198 to 62881 (ORF 53), 62909 to 63862 (ORF 55), 63858 to64271 (ORF 56), 64309 to 66831 (ORF 57), 67266 to 66799 (ORF 58r), 67803to 67273 (ORF 58ar), 67915 to 68607 (ORF 59), 68624 to 70984 (ORF 60),70994 to 72898 (ORF 61), 72938 to 73507 (ORF 62), 73540 to 74211 (ORF63), 76120 to 74207 (ORF 64r), 76749 to 76186 (ORF 65r), 77698 to 76799(ORF 66r), 79343 to 77709 (ORF 67r), 79816 to 79367 (ORF 68r), 80529 to79858 (ORF 69r), 80774 to 80529 (ORF 70r), 82815 to 80788 (ORF 71r),83835 to 82834 (ORF 72r), 83874 to 85583 (ORF 73), 85535 to 84402 (ORF74r), 88096 to 85574 (ORF 75r), 87759 to 88667 (ORF 76), 88920 to 88642(ORF 77r), 91652 to 88938 (ORF 78r), 91667 to 92674 (ORF 79), 93466 to92681 (ORF 80r), 93761 to 93486 (ORF 81r), 94060 to 93788 (ORF 82r),94238 to 94080 (ORF 83r), 94508 to 94242 (ORF 84r), 95571 to 94498 (ORF85r), 96187 to 95600 (ORF 86r), 96202 to 97665 (ORF 87), 97915 to 97643(ORF 88r), 98251 to 99537 (ORF 89), 99537 to 99974 (ORF 90), 100001 to101140 (ORF 91), 101168 to 104650 (ORF 92), 106354 to 104795 (ORF 93r),107947 to 106400 (ORF 94r), 108256 to 107990 ORF 95r), 108719 to 108300(ORF 96r), 109679 to 108738 (ORF 97r), 109861 to 109682 (ORF 98r),110830 to 10033 (ORF 99r), 110208 to 110417 (ORF 100), 110469 to 110651(ORF 100a), 110915 to 111397 (ORF 101), 111419 to 111913 (ORF 102),111949 to 112485 (ORF 103), 112593 to 113450 (ORF 104), 113323 to 112967ORF 105r), 113526 to 114152 (ORF 106), 114199 to 115236 (ORF 107),115353 to 115787 (ORF 108), 115859 o 116551 (ORF 109), 116729 to 117523(ORF 110), 117572 to 117114 (ORF 111r), 117423 to 118085 (ORF 12),118968 to 118375 (ORF 114r), 118508 to 119119 (ORF 115), 119588 to120202 (ORF 116), 120314 to 21231 (ORF 117), 121380 to 123920 (ORF 118),121288 to 122256 (ORF 119), 122350 to 123924 (ORF 120), 123962 to 125566(ORF 121), 125193 to 124591 (ORF 122r), 125689 to 123935 (ORF 123r),123839 to 123297 ORF 123ar), 125652 to 126170 (ORF 124), 126121 to125699 (ORF 125r), 126279 to 127769 (ORF 126), 127851 to 128408 (ORF127), 128520 to 130076 (ORF 128), 130105 to 131700 (ORF 129), 131790 to133283 (ORF 130), 133246 to 133920 (ORF 131), 133972 to 134370 (ORF132), 134418 to 134693 (ORF 133a), 134402 to 134992 (ORF R1), 134853 to134419 (ORF R2r), 135628 to 135897 (ORF R3), 136780 to 137112 ORF R4),and 137558 to 137022 (ORF R5r) of SEQ ID NO:1, which encode for theidentified open reading frames (ORFs) listed in Table 7. ORFs of thisparagraph of which the start position is a larger number than the stopposition are coded by the complementary sequence of SEQ ID NO:1. Thenames of these ORFs end with the letter “r”. The invention also relatesto the complementary sequences of the sequences of this paragraph.

The invention also relates to polynucleotides which encode for the sameamino acid sequence as encoded by the identified ORFs of the previousparagraph. The invention also relates to polynucleotides of at least 15,30 or 100 nucleotides binding under stringent conditions to theidentified ORFs. The invention also relates to polynucleotides whichshow at least 99%, 95% or 90% or 80% sequence homology to the sequencesof the previous paragraph or which are functional variants a sequence ofthe previous paragraph.

A functional variant of a gene, within the meaning of the invention,shall be defined as a gene which is at least 99%, or 95%, or 90%, or 80%homologous to the first gene and which has a similar biological functionas the first gene. A functional variant of a gene can also be a secondgene encoding the same amino acid sequence as does the first gene (or asdoes a functional variant thereof), employing the degeneration of thegenetic code. A functional variant of a gene can also be apolynucleotide comprising the same sequence as has said gene, howeversaid polynucleotide being shorter (i.e., by means of deletions of one orseveral nucleotides at one or both ends of the polynucleotide) or saidpolynucleotide having additional nucleotides at one or both ends of theidentical part of the polynucleotide.

A functional variant of a protein, within the meaning of the invention,shall be defined as another protein which is at least 99%, or 95%, or90%, or 80% homologous to the first protein and which has a similarbiological function as has the original protein.

The invention also relates to recombinant proteins encoded bynucleotides of the invention and parts and fragments of said proteinswhich are at least 5 or 7 or 10 or 30 amino acids long.

The invention also relates to recombinant proteins encoded bynucleotides of the invention and parts and fragments of said proteinswhich are at least 5 or 7 or 10 or 30 amino acids long, said recombinantproteins being attached to a carrier protein or to another carrier.Attaching a protein to a carrier protein can improve or strengthen theimmune response to said protein, thereby enhancing the therapeutic orprophylactic effect of administering said protein to a subject.

The invention also relates to vectors containing polynucleotides of theinvention and cells containing these vectors or polynucleotides of theinvention.

The invention also relates to the use of recombinant proteins andpolynucleotides of the invention, alone or in combination with at leastone other recombinant protein or polynucleotide of the invention for themanufacture of pharmaceutical compositions.

Combinations of recombinant proteins (or polynucleotides) according tothe invention, comprise

-   -   combinations of at least two recombinant proteins encoded by SEQ        ID NO:1 (or combinations of at least two fragments of a        polynucleotide of SEQ ID NO:1),    -   combinations of at least two recombinant proteins encoded by the        same active fragment of the PPVO genome, i.e., two or more        recombinant proteins encoded by the same VVOV of    -   Table 3, Table 4, Table 5, and Table 6 (or combinations of at        least two fragments of the same active fragment (VVOV) of the        PPVO genome),    -   combinations of at least two recombinant proteins, encoded by at        least two distinct active fragments of the PPVO genome, i.e.,        from distinct VVOVs of    -   Table 3, Table 4, Table 5, and Table 6 (or combinations of at        least two fragments of at least two distinct active fragments        (VVOVs) of the PPVO genome), or    -   combinations of at least two distinct recombinant proteins        encoded by ORFs of Table 7 (or combinations of at least two        polynucleotides with the sequence of any of the ORFs listed in        Table 7).

The invention also relates to the use of recombinant viruses comprisingthe Vaccina lister genome and selected fragments of the PPVO genome forthe manufacture of pharmaceutical compositions.

The invention also relates to the use of recombinant proteins andpolynucleotides of the invention for the manufacture of pharmaceuticalcompositions for the treatment of virus related diseases, viralinfections, non-viral infections, proliferative diseases, inflammatorydiseases, allergic diseases, and autoimmune diseases.

Viral infections, within the meaning of the invention, shell beunderstood as diseases associated with viral infections of the human oranimal body, such as hepatitis, papillomatosis, herpes virus infections,liver fibrosis, HIV infections, AIDS, and influenza.

Non-viral infections, within the meaning of the invention, shell beunderstood as diseases associated with non-viral infections of the humanor animal body, such as infections with mycobacteria, mycoplasma,amoeba, and plasmodia.

Proliferative diseases, within the meaning of the invention, shell beunderstood as diseases associated with proliferative disorders, such ascancer, leukemia, warts, tumor diseases, and other skin neoplasms.

Inflammatory diseases, within the meaning of the invention, shell beunderstood as diseases associated with acute or chronic inflammatoryconditions, such as inflammation of the skin or organs, Crohn's disease,COPD, asthma, but also conditions related to the healing of wounds,e.g., Ulcus cruris, and others.

Allergic diseases, within the meaning of the invention, shell beunderstood as comprising both systemic and topical allergies.

Autoimmune diseases within the meaning of the invention, shell beunderstood as comprising systemic lupus erythematosus, Sjogren'ssyndrome, Hashimoto's thyroiditis, rheumatoid arthritis, and juvenilediabetes mellitus, and other autoimmune diseases.

The invention also relates to the use of recombinant viruses comprisinga Vaccinia lister genome and fragments of a PPVO genome for themanufacture of pharmaceutical compositions.

The invention also relates to the use of recombinant viruses comprisinga Vaccinia lister genome and at least one heterologous gene to expressat least one heterologous gene in a subject, e.g., for prophylacticand/or therapeutic purposes.

The invention also relates to the use of a recombinant virusescomprising a Vaccinia lister genome and at least one heterologous genefor gene therapy.

“Gene therapy”, within the meaning of the invention, shall be understoodas the act of administering to a subject polynucleotides (and, ifnecessary, suitable adjuvants or suitable carriers) for the purpose ofobtaining a prophylactic or therapeutic effect in said subject.Typically, the polynucleotides administered are expressed in the subjectand the expressed gene products exert a prophylactic or therapeuticeffect.

The invention also relates to

-   -   (a) a particle-like structure comprising a recombinant        polypeptide encoded by an open reading frame (ORF) of the        polynucleotide of SEQ ID NO:1 or functional variants of said        polypeptides,    -   (b) the use of a particle-like structure of (a) for the        preparation of a medicament,    -   (c) the use of a particle-like structure of (a) for the        preparation of a medicament for the treatment of virus related        diseases, viral infections, non-viral infections, proliferative        diseases, inflammatory diseases, allergic diseases, and/or        autoimmune diseases,    -   (d) pharmaceutical compositions comprising a particle-like        structure of (a), and to    -   (e) pharmaceutical compositions comprising a particle-like        structure of (a) for the treatment of virus related diseases,        viral infections, non-viral infections, proliferative diseases,        inflammatory diseases, allergic diseases, and/or autoimmune        diseases.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows the genomic locations of the DNA fragments constituting theinsertion library. The position of each DNA fragment is shown againstthe KpnI map of PPVO NZ2 (Mercer, et al., Virology (1997) 229:193-200).

EXAMPLES Example 1 Determination of the Integrated PPVO Fragments in theActive VVOVs

DNA preparation from Vaccinia lister/PPVO recombinants was performed asfollows:

BK-KL 3A cells were grown to confluency in 175 cm² flasks (BectonDickson Labware, Heidelberg, Germany). Cells were infected with arecombinant Vaccina lister/PPVO virus (VVOV) of Mercer, et al. (Virology(1997) 229:193-200) at a MOI (multiplicity of infection) of 0.01-0.32and incubated at 37° C. until 100% CPE (cytopathic effect) had beenreached. The infected cells were frozen at −80° C., thawed and processedas follows, with modification to the RNA extraction method of Vilcek, etal. (J. Clin. Microbiol. (1994) 32:2225-2231). Using 2 ml PLG HeavyEppendorf tubes (Eppendorf, Hamburg, Germany) 0.5 ml aliquots ofcellular suspension were incubated with 100 μg Proteinase K (RocheMolecular Biochemicals, Mannheim, Germany) and 50 μl SDS (Sigma-Aldrich,Chemie GmbH, Taufkirchen, Germany) at 56° C. for 25 min 0.5 mlRoti®-Phenol/Chloroform (Carl Roth GmbH, Karlsruhe, Germany) was addedand the tubes were inverted for several times. After centrifugation at12000×g for 10 min, the upper phase was transferred into a fresh tubeand two volumes of ethanol (Merck Eurolab GmbH, Darmstadt, Germany) and1/10 volume of sodium acetate (Sigma-Aldrich, Chemie GmbH, Taufkirchen,Germany) was added. The reagents were mixed several times and stored at−80° C. for 3 h. The tubes were centrifuged at 14000×g for 30 min, thesupernatant was decanted and the pellet was air-dried for 5-10 minFinally the DNA pellet was resuspended in 30 μl nuclease free water andstored at −20° C. until used.

DNA concentration was measured spectrophotometrically on a BioPhotometer6131 (Eppendorf, Hamburg, Germany) at 260/280 nm. The DNA yield ofdifferent sample preparations spanned from 100 ng/ml up to 1 μg/ml.

Polymerase Chain Reaction (PCR) of Terminal Flanking Regions of theIntegrated Fragments in the Vaccinia Lister/PPVO Recombinants wasPerformed as Follows:

Three different PCR amplification systems were used for amplifying theterminal flanking regions. Each reaction mixture of 50 μl contained 100ng-1 μg resuspended DNA and primers (Table 1)) were added in a finalconcentration of 300 nM. Amplifications were carried out on aMastercycler® gradient (Eppendorf, Hamburg, Germany).

The 3-prime flanking region of recombinant VVOV 285 had been analyzedusing 2× Ready-Mix™ PCR Master Mix (1.5 mM MgCl₂) (AB Gene, Hamburg,Germany). 1 μl BSA (MBI Fermentas GmbH, St. Leon-Rot, Germany) was addedto each reaction. Denaturation was performed at 94° C. for 3 min,followed by 30 cycles (94° C. for 30 s, 58.7° C.-65.3° C. for 30 s, 72°C. for 1 mM) and 72° C. for 5 min.

The 5-prime flanking region of the PPVO insert of recombinant VVOV 285,the 3-prime flanking region of VVOV 97, and both terminal flankingregions of VVOV 215, VVOV 243, VVOV 245 were amplified using PfuTurbo®DNA Polymerase (Stratagene, Amsterdam, Netherlands). The reactions weresetup with 2.5 U of enzyme, 1.5 mM MgCl₂ and 200 μM of each dNTP.Denaturation was performed at 94° C. for 3 mM, followed by 30 cycles(94° C. for 30 s, 58.7° C.-65.3° C. for 30 s, 72° C. for 1 mM) and 72°C. for 5 min.

The amplification of the 5-prime flanking region of VVOV 97 and VVOV 82,the 3-prime flanking region of VVOV 96 and VVOV 283 were performed withPlatinium® Pfx DNA Polymerase (Life Technologies GmbH, Karlsruhe,Germany). A reaction of 50 μl contained 1.25 U polymerase, 1-1.5 mMMgCl₂ and 300 μM of each dNTP. Additional use of PCRx Enhancer Solutionwas necessary for amplification of the 5-prime flanking regions of VVOV96 (lx concentrated) and the 3-prime flanking regions of VVOV 82 (2×concentrated). Denaturation was performed at 94° C. for 2 mM, followedby 30 cycles (94° C. for 15 s, 54.6° C.-60.7° C. for 30 s, 68° C. for1-2 mM) and 68° C. for 5-7 min.

18 μl of each amplification product was analyzed by agarose gelelectrophoresis on 1.5-2% SeaKem LE agarose (Biozym, Hessisch Oldendorf,Germany). After staining in a ethidium bromide solution for 20 mM theDNA fragments were visualized on an UV transilluminator UVT-20 M/W(Herolab, Wiesloch, Germany).

The sequence of the amplified DNA-fragments were determined by standardsequencing procedures and compared to the published Vaccinia listerthymidine kinase-sequence and the genome sequence of PPVO NZ2 todetermine exactly the integrated PPVO NZ2 sequences.

TABLE 1 PCR-primers, amplification and sequencing  of the terminal flanking regions of   the integrated fragments in the  Vaccinia lister/PPVO NZ2 recombinants Ampli- Length fied of terminalPrimers used amplifi- region for amplification SEQ cation VVO of NZ2Primer  Sequence ID product V insert name 5′ → 3′ NO: [bp] VVO 5′ VAC-ATTACAGTGATG  2  264 V 215 P11-1 CCTACATGCCG PPVO  GCTGTAGTCGT  3 14r-1GGTCCGGC 3′ PPVO  CTTCCTAGGCT  4  402 4r-2 TCTACCGCACG VAC- CGGTTTACGTT 5 TK-1 GAAATGTCCCAT VVO 5′ VAC- ATTACAGTGATG  2  553 V 245 P11-1CCTACATGCCG PPVO  CTGGCCAACG  6 57-1 ACGCCTTC 3′ PPVO  TCTGGTACCCC  7 321 40-1 TTGCCGG VAC- CGGTTTACGTT  5 TK-1 GAAATGTCCCAT VVO 5′ VAC-ATTACAGTGAT  2  241 V 285 P11-1 GCCTACATGCCG PPVO  GAACCCGCTCT  8 78r-5CGCTCGA 3′ PPVO  GCCGGGCAAGT  9  320 64r-1 GTCTGGTC VAC- CGGTTTACGTTG  5TK-1 AAATGTCCCAT VVO 5′ VAC- ATTACAGTGAT  2  392 V 330 P11-1GCCTACATGCCG PPVO  CTCGAAGTAGC 10 92-1 TGATGTCGCG 3′ PPVO  AGAGCTTTAC 11 462 96r-1 GTAGACTCT CCAAGTGTC VAC- CGGTTTACGTT  5 TK-1 GAAATGTCCCAT VVO5′ VAC- ATACGGAACGGG 12  239 V 96 TK-fwd ACTATGGACG PPVO   GCGGTGGCCATG13 22r-3 TACGTG 3′ PPVO   GGTTGTGGCGA 14 1055 22r-4 TGGTCGG VAC-CGGTTTACGTT  5 TK-1 GAAATGTCCCAT VVO 5′ VAC-  ATACGGAACGGG 12  309 V 97TK-fwd ACTATGGACG PPVO   CTTGATGAGCCG 15 18r-1 GACGCA 3′ PPVO CCGAGTTGGAG 16  318 25r-1 AGGAAGGAGC   VAC- CGGTTTACGTT  5 TK-1GAAATGTCCCAT VVO 5′ VAC- ATTACAGTGAT  2  478 V 243 P11-1 GCCTACATGCCGPPVO  CTGTTGGAGGAT 17 79-1 GAGGTCAAGGA 3′ PPVO  CGTGCTCATGC 18  26971r-1 CTGTGGAC VAC- CGGTTTACGTT 5   TK-1 GAAATGTCCCAT VVO 5′ V 283 3′PPVO  CGACATCCTCA 19  234 92-4 CCTGCAAGAAG VAC- CGGTTTACGTTG  5 TK-1AAATGTCCCAT VVO 5′ VAC- ATACGGAACGG 12  275 V 82 TK-fwd GACTATGGACG  PPVO  TACAGGCAGCC 20 120-1 CGTGACC 3′ PPVO  GCCGTGTGTC 21 1960 R3R4-3ACGTTGATGC   VAC- CGGTTTACGTT  5 TK-1 GAAATGTCCCAT

Example 2 Induction of Interferon Gamma and Tumor Necrosis Factor Alphaby PPVO Gene Products

The 16 recombinants were tested of their ability to induce tumornecrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) in wholeblood cultures.

Whole blood cultures containing blood and RPMI medium (Life TechnologiesGmbH, Karlsruhe, Germany) in the ratio of 1:5 were stimulated with therecombinant viruses. A pure Vaccinia lister and a whole PPVO preparationserved as controls. All preparations were used at a final dilution of1:10. The stimulation for the IFN-7 determination was done together withConcanavalin A (SIGMA, St. Louis, Mo.), because the virus alone does notinduce IFN-γ. Then the cells were incubated for 24 h (TNF-α) and or 72 h(IFN-γ). The cytokine concentration was then determined in the cellculture supernatants by TNF-α or IFN-γ specific ELISA. These time pointswere found to be optimal when the experimental conditions weredetermined using whole PPVO as a control.

It was possible to identify 5 active recombinant viruses (VVOV 96, VVOV97, VVOV 243, VVOV 285, and VVOV 330) that induced both TNF-α and IFN-γsecretion and, thus, could mimic the effect of the whole PPVO. Theresults are depicted in Table 2.

TABLE 2 TNF-α was determined after 24 h stimulation of blood cells withthe recombinant virus or the controls, respectively. IFN-γ wasdetermined after 72 h stimulation of blood cells with the recombinantvirus or the controls. Stimulation was performed in the presence of themitogen ConA. The relative induction in percent of the Vaccinia viruscontrol is shown. Therefore, values greater than 100% are due to theactivity of the PPVO fragments. Active PPVO fragments are in bold. Thedata represent mean values of three different blood donors. RecombinantVirus Clone or Interferon TNF control Induction (%) Induction (%)Vaccinia virus control 100 100 NZ-2 control 2224 264 VVOV 80 200 66 VVOV82 173 65 VVOV 85 209 94 VVOV 86 138 73 VVOV 96 1638 1016 VVOV 97 17131285 VVOV 212 94 62 VVOV 213 192 38 VVOV 215 97 82 VVOV 216 197 71 VVOV243 1446 933 VVOV 245 98 45 VVOV 247 85 74 VVOV 283 115 78 VVOV 285 11281127 VVOV 330 1762 2135

TABLE 3 The recombinant Vaccinia lister/PPVO viruses that induce bothinterferon gamma and TNF-α expression are listed in column 1, thecorresponding PPVO sequence in column 2 and all open reading frames(ORFs) that are completely or partially contained in the recombinant aredepicted in column 3. PPVO NZ2 Sequence PPVO NZ2 ORFs Active recombinant[Bp] that is contained in that are contained in PPVO Vaccinia virus therecombinant the recombinant VVOV 97 24056-33789  18r-25r VVOV 9631003-46845 22r-39 VVOV 285 74804-88576 64r-76 VVOV 243 82324-9250271r-79 VVOV 330 102490-108393   92-96r

Example 3 Local Immunomodulation by PPVO Gene Products in Liver SinusEndothelial Cells (LSEC)

We have established a new cell-based assay system that allows testing ofhepatoprotective properties of recombinant PPVO proteins expressed indifferent systems (e.g., Vaccinia virus). This assay system uses primarymurine liver cells, which play the central role in deciding whetherimmunity or tolerance is induced in the liver, the LSEC. The uniqueability of LSEC to present exogenous antigens to CD8+ T cells on MHCclass I molecules allows immune surveillance of hepatocytes as viralantigens released by infected hepatocytes are likely to be taken by LSECand presented to cells of the immune system. The new assay allows tomeasure the ability of LSEC to interact antigen-specifically with CD8+ Tcells, that are responsible for tissue destruction in necroinflammatoryhepatitis.

Pure populations of LSEC are isolated from murine liver by a stepwiseprocedure of portal-vein perfusion with collagenase A (0.05%),mechanical dispersion and further enzymatic digestion in a rotatorywaterbath for 40 min at 37° C. (245 rpm), gradient centrifugation(metrizamide 1.089 g/cm³) and centrifugal elutriation using a BeckmanAvanti J25I centrifuge equipped with a JE-6B rotor and a standardelutriation chamber. LSEC cell populations isolated by this method aretypically around 95-99% pure as measured by uptake of endothelial cellspecific substrate (acetylated low density lipoprotein). LSEC wereseeded onto collagen type I coated 24 well tissue culture plates at adensity of 100.000 cells per well and were further cultured inDulbecco's modified Eagle Medium supplemented with 5% fetal calf serum(specially tested not to interfere with the assay system) and 2%glutamine. Three days after isolation, when LSEC gained a post mitoticand quiescent state, we tested for the ability of LSEC to presentsoluble ovalbumin to (ovalbumin-specific) CD8+ T cells. LSEC wereincubated with 1 μM of ovalbumin for three hours (antigen dose and timewere previously shown to be optimal for testing of substances suspectedto influence antigen-presentation), washed and incubated with a CD8+ Tcell hybridoma (200.000 cells/well) that recognizes the peptide SIINFEKL(SEQ ID NO:320). SIINFEKL (SEQ ID NO:320) is recognized in a H2b contextand directly binds on the MHC-I molecules. Therefore, it has not to beprocessed by the cell. This allows to differentiate between accessoryfunctions of LSEC (such as MHC-I expression) and antigen-processingfunction.

The extent of CD8+ T cell activation was measured by determining theextent of IL-2 release from T cells by specific sandwich ELISA.

Using Vaccinia virus expressed recombinant proteins derived from PPVO wehave been able to attribute hepatoprotective activity to individualclones. To be able to compare different clones directly with respect totheir ability to influence cross-presentation by LSEC, we used equalamounts of “infectious units”.

We found that LSEC cross-present exogenous ovalbumin very efficiently onMHC class I molecules (k^(b)) to CD8+ T cells. To our surprise we foundif LSEC were incubated with several recombinant PPVO proteins weobserved subsequently a potent downregulation of cross-presentation bymore than 60% compared to the mock-treated control that includes all butthe active ingredient. Several regions within the genome of PPVO haveimmunoregulatory properties. Especially the region termed 82 (43%reduction) which is located at the 3′ end of the genome appears to beresponsible for the overall effect of PPVO on cross-presentation byLSEC. Further regions (VVOV 215, VVOV 212, VVOV 247 and VVOV 86) bearfurther immunoregulatory potential, although to a lesser degree (around30% reduction in cross-presentation). It further appears that genescoding for proteins that downregulate cross-presentation are arranged inclusters. It is of interest to note that we identified two gene clusterscoding for proteins that improved cross-presentation (VVOV 330, VVOV283, VVOV 285, VVOV 97, and VVOV 96). However, for unknown reasons thedownregulatory effect of the proteins mentioned above is dominant in theactivity of PPVO on cross-presentation.

Our results strongly suggest that PPVO contains a mixture of differentproteins that in a complementary way work to eliminate hepatocytes fromhepatitis B virus while conserving hepatic integrity and avoiding longlasting damage secondary to hepatic fibrosis. As PPVO contains a genewith high homology to the anti-inflammatory agent IL-10 (located in the5-prime region of the genome) we wondered whether the potentdownregulatory effect of the clone 82 was due to expression of ovineIL-10. This assumes that there is cross-reactivity between murine andovine IL-10 at the level of receptor recognition. We have been unable todemonstrate involvement of ovine IL-10 on the immunoregulatory potentialof PPVO. Recombinant murine IL-10 did not show any influence oncross-presentation through LSEC and several monoclonal antibodies tomurine and human IL-10 did not influence PPVO mediated downregulation ofcross-presentation. We conclude that the immunoregulatory component ofPPVO is probably not IL-10 but a new, so far not identified mediator.The data for the MHC-I cross-presentation—down-modulating recombinantvirus are depicted in Table 4, those for the MHC-Icross-presentation—stimulating recombinant viruses in Table 5.

TABLE 4 The recombinant Vaccinia lister/PPVO virus that down-modulatesthe MHC-I cross presentation is designated in column 1, thecorresponding PPVO sequence in column 2 and all open reading frames(ORFs) that are completely or partially contained in the recombinant aredepicted in column 3. PPVO NZ2 Sequence PPVO NZ2 ORFs Active recombinant[Bp] that is contained in that are contained in PPVO Vaccinia virus therecombinant the recombinant VVOV 82 122616-136025 120-R3

TABLE 5 The recombinant Vaccinia lister/PPVO viruses that stimulate theMHC-I cross presentation are designated in column 1, the correspondingPPVO sequence in column 2 and all open reading frames (ORFs) that arecompletely or partially contained in the recombinant are depicted incolumn 3. PPVO NZ2-Sequence PPVO NZ2-ORFs Active recombinant [Bp] thatis contained in that are contained in PPVO Vaccinia virus therecombinant the recombinant VVOV 97 24056-33789  18r-25r VVOV 9631003-46845 22r-39 VVOV 285 74804-88576 64r-76 VVOV 283   89,4-10348378r-92 VVOV 330 102490-108393   92-96r

Example 4 Determination of the Immunostimulatory Activity of theVaccinia Lister/PPVO Recombinants in the Aujeszky Mouse Model

We also tested the activity of recombinant Vaccinia lister/PPVONZ2-viruses in the Aujeszky mouse model, a lethal challenge model ofacute Suid Herpesvirus 1 disease for determining the activity of variousimmunostimulators (e.g., Baypamun®, CpG oligonucleotides).

a) Conditions Employed for the Mice

The NMRI mice (outbreed strain HdsWin:NMRI; female; weight: 18-20 g;obtained via Harlan/Winkelmann, Borchen, Germany) were kept inautoclavable polycarbonate crates lined with sawdust in an S2 isolationstall at 20-22° C. (atmospheric humidity: 50-60%) and subjected to anartificial day/night rhythm (illumination from 6:30 h to 18:30 h anddarkness from 18:30 h to 6:30 h). They had free access to feed andwater.

b) Challenge Model

Groups of mice consisting of 10 mice per group were used for the tests.All of the animals in one group were given the same test substance.

After the mice were supplied they were kept in the animal stall for 2-3days. Then the Vaccinia lister/PPVO NZ2 recombinants were diluted withPBS (Life Technologies GmbH, Karlsruhe, Germany) to a titer equivalentof approx. 10⁸ TCID₅₀/ml and thermally inactivated (twice for one hourat 58° C.). Of these solutions 0.2 ml was administered per mouseintraperitoneally.

24 hours after the treatment the mice were infected with thepseudorabies virus of the Hannover H2 strain by intraperitonealadministration. For this purpose the virus was diluted in PBS to a testtiter of approx. 10⁴ TCID₅₀/ml and 0.2 ml of this suspension wasadministered.

As a negative control one group of mice was treated with PBS and theninfected. The mice in this group died 3-8 days after infection. A largeproportion of the mice treated the Vaccinia lister/PPVO NZ2 recombinantsVVOV 215, VVOV 245, VVOV 285 or VVOV 330 survived infection with thepseudorabies virus. 10 days after the infection with the virus the testwas ended.

The level of induced immunostimulation was determined by comparing thenumber of dead mice in the PBS control group with the number of deadmice in the test groups and was quantified by the efficacy index (EI).This index indicates the percentage proportion of mice protected againstthe lethal effects of the Aujeszky virus infection through immunestimulation by the substance to be tested. It is calculated by means ofthe following formula:

EI=(b−a)/b×100,

where b is the percentage proportion of the dead mice in the controlgroup and a the percentage proportion of the dead mice in the testgroup.

A chi-square test was used for the statistical evaluation. This testreveals the minimum activity indices indicating a significant differencebetween the mortality rate of those mice treated with the test substanceand those treated with PBS. Activity indices of ≧60% are significantwhere at least 5 of the mice used in tests with n=6 mice per group inthe PBS control group and at least 7 of the mice used in tests with n=10in the PBS control group do not survive the infection with the Aujeszkyvirus.

Altogether 3 separate tests were carried out in each case. The testingof Vaccinia lister/PPVO NZ2 recombinants in the Aujeszky mouse modelshows the following:

Surprisingly, after the treatment of the mice with the Vaccinialister/PPVO NZ2 recombinants VVOV 215, VVOV 245, VVOV 285 or VVOV 330the average activity indices of higher than 60% demonstratedimmunostimulation. By contrast all of the other Vaccinia lister/PPVO NZ2recombinants were ineffective. The data is summarized in Table 6.

TABLE 6 The recombinant Vaccinia lister/PPVO viruses that protected micefrom herpesvirus induced death are designated in column 1, thecorresponding PPVO sequence in column 2 and all open reading frames(ORFs) that are completely or partially contained in the recombinant aredepicted in column 3. PPVO NZ2-Sequence PPVO NZ2 ORFs Active recombinant[Bp] that is contained in that are contained in PPVO Vaccinia virus therecombinant the recombinant VVOV 215 10264-20003  4r-14r VVOV 24547952-66263 40r-57 VVOV 285 74804-88576 64r-76 VVOV 330 102490-108393  92-96r

TABLE 7 Sequences of the Parapox ovis open reading frames. ORFs thenames of which end with “r” are encoded on the complementaryDNA strand. Base pair positions in the “from” and “to”column are relative to SEQ ID NO: 1. SEQ SEQ ID ID ORF from to N-term NOC-term NO Comment  L1      3    539 IRGFAG  22 PQKVFRL  23long termal repeat  (LTR)-protein, retroviral  pseudoprotease  L2r   781    449 MSEGGRL  24  LLGLLFP  25 LTR-protein,  retroviralpseudoprotease  L3r   1933   1664 MTVHPPK  26 VLPPNSL  27  LTR-protein, retroviral pseudoprotease  L4r   3269   2790  MHPSPRR  28 PVSHPFL   29 LTR-protein,  retroviral pseudoprotease  L5   2799   3851 MGDREGE  30 FEDGVKC  31 LTR-protein,  retroviral pseudoprotease  L6   2962   3753 MCTVATF  32 GAPRAGW  33  LTR-protein,  similar to 134r, retroviral pseudoprotease  L7r   3784   3122 MTPTSRE  34  ARTAPPR  35 LTR-protein, retroviral pseudoprotease  L8r   4341   4129  MPGEGQY  36  NGGLGKI  37LTR-protein,  retroviral pseudoprotease   1ar   4904   4428 MEFCHTE  38DTAWYIS  39 dUTPase   1r   6517   4970 MLSRESV  40 RAMLTRP  41 homolog of G1L in NZ2,  Ankyrin-repeats   2r   8042   6684  MFFWFWC  42 SGEGVPV  43   3r   9989   8070  MLGFWGK  44  VLPSVSR  45 involved in maturation  of EEV (Extracellular Enveloped Virions)   4r 11195  10062 MWPFSSI  46 EFCKPIN  47  Phospholipase D-type enzyme   5r 11493  11227 MLIYGPR  48 RLLKDFP  49 homolog of B3L in NZ2   6  11802 12038 MGVVMCG  50 APAGVTE  51   7r  12358  12080 MPVKVKQ  52  ASREFIV 53 ubiquitination  protein with RING- finger-motiv (relatedto yeast proteins APC11  and HRT1)   8r  13980  12364  MEEELTR  54 SPMVVFN  55 no Vaccinia  virus homolog   9ar  14826  14053  MIRIGGG  56 DNMRVDD  57  10  15080  15394 MDGGVHK  58 EQMCRRQ  59 virion coreDNA-binding phosphoprotein  11r  16838  15423 MAPPVIE  60 AKNVITH  61polyA polymerase  12r  19021  16847  MLQLLKR  62 NNRGFRK  63  13r  19704 19156 MACECAS  64 NNCGISF   65 interferon resistance protein, homology to mammalian PACT (protein activator of theinterferon-induced  protein kinase) also called PRKRA (dsRNAdependent activator of Interferon-induced  protein kinase),13r-protein contains a  dsRBD motiv (double- stranded RNA bindingdomain) and a ′DRADA′- domain that is typical for RNA-editing enzymes) 14r  20314  19736 MDEDRLR  66 KKGKPKS  67 RNA polymerase  15  20401 22101 MDFVRRK  68 VVLQGRA  69  16  22125  22940 MVDSGTH  70 PENVVLL  71  17  23003  23866 MASYISG  72 RTHTVYV  73  18r  26908  23873 MLFEMEL 74 SKPVFTG   75 DNA polymerase  19  26926  27213 MEPRFWG  76 AKVRPLV  77 distant homolog of  the ERV1/ALR-protein- family (ERV1: yeast protein, Essential for Respiration and Vegatative growth, ALR:mammalian protein, Aug- menter of Liver Regeneration)  20r  27626  27216MEAINVF  78 RAYEGML   79  21r  29754  27616 MLLYPKK  80 LLGDGGD  81related to 12r  22r  32217  29800 MLIRTTD  82 EAQNMQN   83  23r  33380 32418 MEDERLI  84 PSPCGGE  85  24r  33602  33393 MDKLYTG  86 FHYLKLV 87  25r  34466  33612 MKRAVSK  88 LEAPFNI   89 DNA binding phosphoprotein  26r  34735  34502 MESRDLG  90 LNARRQN   91  27r  35905 34739 MNHFFKQ  92 RSLYTVL  93  28r  37194  35905 MDKYTDL  94 PEKPAAP 95 core protein  29  37200  39248 MENHLPD  96 IEAEPPF   97 RNA helicase 30r  41037  39229 MIVLENG  98 RMGARPR  99 Zn-protease,  involved in virion morphogenesis  31  41374  42066 MTFRELI 100 DSMASRS101 late transcription  factor  32r  42336  41731 MRGHPAH 102 VAPREEL103  33r  42407  41997 MASDASP 104 QPSSSRR 105 Glutaredoxin- like enzyme 34  42410  43765 MGIKNLK 106 PRLLKLR 107  35  43770  43958 MVFPIVC 108LPMLDIS 109 RNA polymerase  36  43980  44534 MREFGLA 110 AEPPWLV  111 37r  45727  44537 MESSKQA 112 TRAPPLF  113 core virion protein precursor  38  45760  46557 MTLRIKL 114 DRSLSCD  115late transcription  factor  39  46567   47568 MGGSVSL 116 YLLIVWL 117 40  47572  48303 MGAAASI  118 TEFPPSV  119 virion protein, related to vaccinia F9L  41  48352  48621 MVRRVLL 120 LCLFSMD 121  42r 49887  48634 MEEKRGR 122 ARAMVCL  123  43  49917  50693 MTNLLSL 124TGAEAAP 125 core protein,  DNA binding domain  44  50719  51102 MAAPTTP126 VDVLGGR 127  44a  51059  51511 MDHEKYV 128 ATLSPGL 129  45  51584 52591 MEGVEMD 130 RPLRGGK 131 polyA polymerase  46  52509  53066MNRHNTR 132 SVSVVLD 133 RNA polymerase  47r  53523  53023 MFFRRRA 134GRRPPRP 135  48  53607  57473 MSVVARV 136 EAAEEEF 137 RNA polymerase chain 1  49r  58070  57528 MGDKSEW 138 FVCDSPS 139 tyrosine phosphatase 50  57700   58662 MAAAPLR 140 ATSGVLT  141  51r  59674  58673 MDPPEIT142 LLVTAIV  143 immunodominant  envelope protein  52r  62089  59678MDSRESI 144 YMINFNN  145 RNA polymerase- associated trans-cription specificity  factor (also called  RAP94)  53  62198  62881MSSWRLK 146 KAAACKK 147 late transcription  factor  55  62909  63862MRALHLS 148 NSEQVNG 149 topoisomerase I  56  63858   64271 MDEALRV 150FIRAAVA 151  57  64309   66831 MDAPSLD 152 LYVFSKR 153 mRNA capping enzyme  58r  67266  66799 MEPSAMR 154 DVQHVDL 155 virion protein  58ar  67803  67273 MAGFSQS 156 TTCVPPQ 157  59  67915  68607 MATPANA 158FSFYSEN  159 Uracil DNA  glycosylase  60  68624  70984 MAAPICD 160IEDVENK 161 ATPase, involved  in DNA replication  61  70994  72898MNSDVIK 162 EVSVVNI 163 early transcription  factor  62  72938   73507MSTFRQT 164 ASPAAKN  165 RNA polymerase  63  73540  74211 MRTYTSL 166WGAAVTR 167 NTP pyrophos- phohydrolase  64r  76120   74207 MTSAHAA 168VDPASIA 169 virion NTPase  65r  76749  76186 MEGRARF 170 RFCNYCP 171 66r  77698  76799 MKTDCAS 172 KLKLLLQ 173 mRNA capping enzyme  67r 79343   77709 MNNSVVS 174 AEKVTAQ 175 rifampicin resistance, virion membrane  68r  79816  79367 MKRIALS 176 MALKSLI 177late transactivator  protein  69r  80529   79858 MNLRMCG 178 AACSLDL 179late transactivator  protein  70r  80774   80529 MGDNVWF  180 VLGLEQA181 thioredoxin-like  protein  71r  82815   80788 MESPACA  182  NMCDVLC183 major core protein  72r  83835  82834 MDLRRRF  184  VDNTGTS 185core protein  73  83874  85583 MEESVAV  186 LLNYGCG 187 RNA-polymerase 74r  85535  84402 MDRLRTC  188 AEAAESA  189  75r  88096   85574MVSVMRK  190 QEFYPQP 191 early transcription  factor  76  87759  88667MFQPVPD  192 SACRASP 193  77r  88920  88642 MRPCYVT  194 TRGTQTG 195 78r  91652  88938 MTAPNVH  196 AVSFDSE 197 major core protein  79 91667  92674 MTAVPVT  198 VRKLNLI 199  80r  93466  92681 MASEKMA 200DLDGGMC 201 virion protein  81r  93761   93486 MGLLDAL  202 RFSAASS 203virion membrane  protein  82r  94060  93788 MDIFETL  204 DIELTAR 205virion membrane  protein  83r  94238  94080 MVSDYDP  206 HFVHSVI 207 84r  94508  94242 MFLDSDT 208 DMPFSVV 209  85r  95571  94498 MGDTVSK210 KTINVSR 211  86r  96187  95600 MESYFSY  212 EDLFFAE  213virion membrane  protein  87  96202  97665 MFGGVQV  214 GRDLAAV 215RNA helicase  88r  97915  97643 MSAVKAK  216 PLRDLAR 217Zn-finger protein  89  98251  99537 MTSESDL  218 AIARAQP 219DNA polymerase  processivity factor  90  99537   99974 MIVAAFD 220 NYVLRTN  221  91 100001 101140 MLALFEF 222 LKELLGP 223 intermediate transcription factor  92 101168 104650 MEQALGY 224 SLFSPED 225RNA polymerase b-chain  93r 106354 104795 MESDNAL 226 GQHAAIW 227A-type inclusion  body/Fusion peptide  94r 107947 106400 MEKLVSD 228GRSGAIW  229 A-type inclusion body/Fusion peptide  95r 108256 107990MDENDGE 230 QTGYSRY 231 viral fusion  protein  96r 108719 108300 MDAVSAL232 LFLKSIL  233    97r 109679 108738 MADAPLV 234 RELRANE 235RNA polymerase  subunit  98r 109861 1109682 MEEDLNE 236 MGQASSA 237  99r110830 110033 MDVVQEV 238 ADSDGGN 239 ATPase 100 110208 110417 MRSWFWQ 240 PLTGMCL 241 100a  110469 110651 MRPKSVG 242 SGHTKPS 243 101 110915111397 MAHNTFE 244 KYFCVSD 245 enveloped virion  glycoprotein 102 111419111913 MGCCKVP 246 CMKEMHG 247 enveloped virion  glycoprotein 103 111949112485 MSRLQIL 248 RKLDVPI 249 104 112593 113450 MKAVLLL 250 LNLNPGN 251GM-CSF/IL-2  inhibition factor 105r  113323 112967 MHASLSS 252 DETLTYR253 106 113526 114152 MEVLVII 254 GEFFYDE 255 107 114199 115236 MPLFRKL256 RDALDGL 257 108 115353 115787 MACFIEL 258 TTFSSSE 259 109 115859116551 MSSSSSE 260 TTGTSTS 261 TT 110 116729 117523 MACLRVF 262 CSMQTAR263 GM-CSF/IL-2  inhibition factor 111r  117572 117114 MAIAHTT 264FRFRTPG 265 112 117423 118085 MAATIQI 266 KRDGYSR 267 114r  118968118375 MEGLMPK 268 RPISVQK 269 115 118508 119119 MDSRRLA 270 LGDSDSD 271116 119588 120202 MRLILAL 272 PQMMRIG 273 117 120314 121231 MAGFLGA 274CKVEEVL 275 118 121380 123920 MHLHKDP 276 LAFPSLA 277 119 121288 122256MANRLVF 278 RPMEIDG 279 120 122350 123924 MENNDGN 280 RFLPSHK 281related to 1r/G1L  with Ankyrin- repeats 121 123962 125566 MDPAGQR 282CSETDRW 283 122r  125193 124591 MSSSAAA 284 IAPDSRM 285 123r  125689123935 MTAEASI 286 DPVYHKK 287 123ar 123839 123297 MPRTTSG 288 REQTEGL289 124 125652 126170 MANREEI 290 VRVLRRT 291 125r  126121 125699MTAPTPR 292 AAYSLAR 293 126 126279 127769 MADEREA 294 LACAMRK 295related to 1r/G1L  with Ankyrin-repeats 127 127851 128408 MSKNKIL 296SYMTTKM 297 sheep-like  Interleukin 10 128 128520 130076 MLTRCYI 298RASGLAE 299 related to 1r/G1L  wih Ankyrin-repeats 129 130105 131700MVGFDRR 300 CGRRAPE 301 related to 1r/G1L,  with Ankyrin-repeats(NT slightly changed) 130 131790 133283 MILARAG 302 PDAAALS 303 Kinase131 133246 133920 MPPRTPP 304 RPAALRA 305 132 133972 134370 MKLLVGI  306RPPRRRR 307 homolog to the  sheep VEGF (Vascular EndothelialGrowth Factor) 133a  134418 134693 MRKKAPR 308  ARTAPPR 309corresponds to L7r R1 134402 134992 MMRSGHA 310 RMHRSEL  311LTR-protein (corresponds  to L4r), retroviral pseudoprotease R2r 134853134419 MCTVATF  312  SVAPSSA 313 LTR-protein (corresponds to L6, 134r), retroviral pseudoprotease R3 135628 135897 MTVHPPK  314VLPPNSL 315 LTR-protein (corresponds  to L3r), retroviral pseudoproteaseR4 136780 137112 MSEGGRL  316 LLGLLFP 317 LTR-protein (corresponds to L2r), retroviral pseudoprotease R5r 137558 137022 IRGFAGG 318 PQKVFRL319 LTR-protein (corresponds to L1r), retroviral pseudoprotease

1. A method for inducing an immunomodulatory activity in a subject,consisting of administering to the subject an individual recombinantprotein encoded by a polynucleotide selected from the group consistingof: (i) a polynucleotide having the sequence consisting of nucleotideresidues 31003 to 46845 of SEQ ID NO:1 (PPVO insert of VVOV 96), (ii) apolynucleotide having the sequence consisting of nucleotide residues24056 to 33789 of SEQ ID NO:1 (PPVO insert of VVOV 97), (iii) apolynucleotide having the sequence consisting of nucleotide residues82324 to 92502 of SEQ ID NO:1 (PPVO insert of VVOV 243), (iv) apolynucleotide having the sequence consisting of nucleotide residues74804 to 88576 of SEQ ID NO:1 (PPVO insert of VVOV 285), and (v) apolynucleotide having the sequence consisting of nucleotide residues102490 to 108393 of SEQ ID NO:1 (PPVO insert of VVOV 330).
 2. The methodof claim 1, wherein the immunomodulatory activity results in theinduction of interferon-gamma.
 3. The method of claim 1, wherein theimmunomodulatory activity results in the induction of tumor necrosisfactor-alpha.
 4. The method of claim 1, wherein the immunomodulatoryactivity results in the stimulation of MHC-I cross-presentation.
 5. Themethod of claim 1, wherein the recombinant protein is attached to or isa part of a structure selected from the group consisting of:particle-like structure, fusion protein, protein coated particle, andvirus-like particle.
 6. A method for inducing an immunomodulatoryactivity in a subject, consisting of administering to the subject anindividual recombinant virus containing a recombinant protein encoded bya polynucleotide selected from the group consisting of: (i) apolynucleotide having the sequence consisting of nucleotide residues31003 to 46845 of SEQ ID NO:1 (PPVO insert of VVOV 96), (ii) apolynucleotide having the sequence consisting of nucleotide residues24056 to 33789 of SEQ ID NO:1 (PPVO insert of VVOV 97), (iii) apolynucleotide having the sequence consisting of nucleotide residues82324 to 92502 of SEQ ID NO:1 (PPVO insert of VVOV 243), (iv) apolynucleotide having the sequence consisting of nucleotide residues74804 to 88576 of SEQ ID NO:1 (PPVO insert of VVOV 285), and (v) apolynucleotide having the sequence consisting of nucleotide residues102490 to 108393 of SEQ ID NO:1 (PPVO insert of VVOV 330).
 7. The methodof claim 6 wherein the immunomodulatory activity results in theinduction of interferon-gamma.
 8. The method of claim 6 wherein theimmunomodulatory activity results in the induction of tumor necrosisfactor-alpha.
 9. The method of claim 6 wherein the immunomodulatoryactivity results in the stimulation of MHC-I cross-presentation.